Functional characterization of the SiFPGS2 gene of foxtail millet in folate accumulation and root development

The folate content of foxtail millet ( Setaria italica ) is higher than that of other major crops. Polyglutamylation of folate can enhance its compartmentalization, reduce its transport, increase its affinities with enzymes, and increase the stability of the folate pool in Arabidopsis . The polyglutamylation reaction is catalyzed by folylpolyglutamate synthetases (FPGSs). However, the function of FPGS in foxtail millet remains unclear. In this study, we identified the Setaria italica FPGS2 ( SiFPGS 2) gene function in foxtail millet. Phylogenetic tree, protein function domain, and docking analyses results revealed that SiFPGS2 belongs to the FPGS subfamily, possesses the tetrahydrofolypolyglutamate synthetase domain, and can bind tetrahydrofolate (THF) as a substrate. Subcellular localization predicted that SiFPGS2 was localized in the chloroplasts. Cis-acting element prediction analysis indicated the presence of meristem expression motif and root specific motif in the SiFPGS2 promoter. Overexpression of SiFPGS2 in Arabidopsis not only increased the folate content, but also increased the root length. Propidium iodide staining revealed that the increased root length was due to the promotion of cell division in the root apical meristem zone. SiFPGS2 catalyzes the polyglutamylation of THF and leads to an increase in folate content and root length by promoting root apical meristem zone cell division. SiFPGS2 can be used as a candidate gene for the biofortification of folate in Gramineae crops via genetic engineering.